Connecting Metabolism to Mastitis: Hyperketonemia Impaired Mammary Gland Defenses During a Streptococcus uberis Challenge in Dairy Cattle.

ß-hydroxybutyrate (BHB) has been associated with disease incidence in early lactation dairy cattle, but such associations do not demonstrate causation. Therefore, the objective of this study was to examine the effects of BHB during an intramammary Streptococcus uberis challenge. A secondary objective was to elucidate the mechanisms behind BHB effects on cytokine transcript abundance using the RAW 264.7 cell line. Late lactation multiparous dairy cows (n = 12) were continuously infused intravenously with either BHB to induce hyperketonemia (target concentration: 1.8 mM) or with saline (CON) for 72 h during a S. uberis intramammary challenge. Body temperature, dry matter intake (DMI), milk production, and milk S. uberis cfu were measured daily until one week post-challenge. Blood samples were collected during infusion to assess changes in metabolism (glucose, insulin, glucagon, NEFA, and cortisol) and systemic inflammation (IL-1ß and SAA). Mammary biopsies were conducted at 72 h post-challenge to assess transcript abundance of inflammation-associated genes. BHB-infused cows exhibited a delayed febrile response, noted by a lesser vaginal temperature during the final day of infusion, followed by a greater vaginal temperature 6 d post-challenge. Consequently, BHB-infused cows had greater S. uberis cfu on d 4, 6, and 7 as compared to CON. Accordingly, BHB-infused cows consumed less DM, produced less milk, had reduced blood glucose, and had increased cortisol concentrations, however, no effects were seen on other systemic parameters or transcript abundance of inflammation-related genes in mammary tissue. To elucidate mechanisms behind the impaired immune defenses, RAW 264.7 cells were transfected with a GPR109A siRNA for 24 h and then treated with or without 1.8 mM BHB and challenged or left unchallenged with S. uberis for an additional 3 h. Transfection with siRNA reduced Gpr109a by 75%. Although BHB treatment did not significantly increase Il10, GPR109A knockdown as compared to the scrambled control reduced Il10 by 90% in S. uberis challenged macrophages treated with BHB, suggesting that macrophage immune responses to S. uberis can be altered via a GPR109A-dependent mechanism. Taken together, these data suggest that BHB altered the immune response promoting tolerance toward S. uberis rather than resistance.

Palmitic Acid and ß-Hydroxybutyrate Induce Inflammatory Responses in Bovine Endometrial Cells by Activating Oxidative Stress-Mediated NF-κB Signaling.

Ketosis is a nutritional metabolic disease in dairy cows, and researches indicated that ketonic cows always accompany reproductive problems. When ketosis occurs, the levels of non-esterified fatty acids (NEFAs) and ß-hydroxybutyrate (BHBA) in the blood increase significantly. Palmitic acid (PA) is a main component of saturated fatty acids composing NEFA. The aim of this study was to investigate whether high levels of PA and BHBA induce inflammatory responses and regulatory mechanisms in bovine endometrial cells (BEND). Using an enzyme-linked immunosorbent assay, quantitative real-time PCR, and western blotting, we evaluated oxidative stress, pro-inflammatory factors, and the nuclear factor (NF)-κB pathway in cultured BEND cells treated with different concentrations of PA, BHBA, pyrrolidinedithiocarbamate (PDTC, an NF-κB pathway inhibitor), and N-acetylcysteine (NAC, an antioxidant). The content of malondialdehyde was significantly higher, the content of glutathione was lower, and antioxidant activity-glutathione peroxidase, superoxide dismutase, catalase, and total antioxidant capacity-was lower in treated cells compared with control cells. PA- and BHBA-induced oxidative stress activated the NF-κB signaling pathway and upregulated the release of pro-inflammatory factors. Moreover, PA- and BHBA-induced activation of NF-κB-mediated inflammatory responses was inhibited by PDTC and NAC. High concentrations of PA and BHBA induce inflammatory responses in BEND cells by activating oxidative stress-mediated NF-κB signaling.

Potential prophylactic effect of recombinant bovine somatotropin (rbST) in sheep with experimentally induced hyperketonemia.

Hyperketonemia in sheep is associated with a disease known as pregnancy toxemia. The purposes of this study were to (1) induce hyperketonemia by infusion with D-beta-hydroxybutyrate (D-BHB) in eighteen healthy, non-pregnant, non-lactating ewes, (2) to evaluate the metabolic pathways in which recombinant bovine somatotropin (rbST) may be involved, and (3) to evaluate the possible benefits of preventive rbST therapy on the metabolic profiles of the sheep. The sheep were intravenously infused with a D-BHB solution over 2 h. Three days prior to the infusion, one group (n = 9) was injected with a single dose of 160 mg rbST, and the other group (n = 9) was injected with saline as a control. Blood samples were collected and metabolic profiles were analyzed every 20 min during the infusion and at 15, 30, 60, 120, 180, and 240 min post-infusion. Hyperketonemia was successfully achieved, demonstrated by serum BHB concentrations of 3.9 and 3.0 mmol/L in the rbST and control groups, respectively, without the presence of clinical signs. An increase in peripheral insulin resistance was observed after infusion but not during the infusion, in the rbST group. Higher glucose concentrations were observed in the rbST group post-infusion, suggesting a positive impact of D-BHB on gluconeogenesis. Our data suggest that prophylactic treatment with rbST may be useful in sheep to reduce the risk of PT.

Bicarbonate correction of ketoacidosis alters host-pathogen interactions and alleviates mucormycosis.

Patients with diabetic ketoacidosis (DKA) are uniquely predisposed to mucormycosis, an angioinvasive fungal infection with high mortality. Previously, we demonstrated that Rhizopus invades the endothelium via binding of fungal CotH proteins to the host receptor GRP78. Here, we report that surface expression of GRP78 is increased in endothelial cells exposed to physiological concentrations of ß-hydroxy butyrate (BHB), glucose, and iron that are similar to those found in DKA patients. Additionally, expression of R. oryzae CotH was increased within hours of incubation with DKA-associated concentrations of BHB, glucose, and iron, augmenting the ability of R. oryzae to invade and subsequently damage endothelial cells in vitro. BHB exposure also increased fungal growth and attenuated R. oryzae neutrophil-mediated damage. Further, mice given BHB developed clinical acidosis and became extremely susceptible to mucormycosis, but not aspergillosis, while sodium bicarbonate reversed this susceptibility. BHB-related acidosis exerted a direct effect on both GRP78 and CotH expression, an effect not seen with lactic acidosis. However, BHB also indirectly compromised the ability of transferrin to chelate iron, as iron chelation combined with sodium bicarbonate completely protected endothelial cells from Rhizopus-mediated invasion and damage. Our results dissect the pathogenesis of mucormycosis during ketoacidosis and reinforce the importance of careful metabolic control of the acidosis to prevent and manage this infection.

Hyperketonemia (acetoacetate) upregulates NADPH oxidase 4 and elevates oxidative stress, ICAM-1, and monocyte adhesivity in endothelial cells.

BACKGROUND/AIMS: The incidence of developing microvascular dysfunction is significantly higher in type 1 diabetic (T1D) patients. Hyperketonemia (acetoacetate, ß-hydroxybutyrate) is frequently found along with hyperglycemia in T1D. Whether hyperketonemia per se contributes to the excess oxidative stress and cellular injury observed in T1D is not known. METHODS: HUVEC were treated with ketones in the presence or absence of high glucose for 24 h. NOX4 siRNA was used to specifically knockdown NOX4 expression in HUVEC. RESULTS: Ketones alone or in combination with high glucose treatment cause a significant increase in oxidative stress, ICAM-1, and monocyte adhesivity to HUVEC. Using an antisense approach, we show that ketone induced increases in ROS, ICAM-1 expression, and monocyte adhesion in endothelial cells were prevented in NOX4 knockdown cells. CONCLUSION: This study reports that elevated levels of ketones upregulate NOX, contributing to increased oxidative stress, ICAM-1 levels, and cellular dysfunction. This provides a novel biochemical mechanism that elucidates the role of hyperketonemia in the excess cellular injury in T1D. New drugs targeting inhibition of NOX seems promising in preventing higher risk of complications associated with T1D.

Pathway of programmed cell death and oxidative stress induced by ß-hydroxybutyrate in dairy cow abomasum smooth muscle cells and in mouse gastric smooth muscle.

The administration of exogenous ß-hydroxybutyrate (ß-HB), as well as fasting and caloric restriction, is a condition associated with ß-HB abundance and decreased appetite in animals. Increased ß-HB and decreased appetite exist simultaneously in some diseases, such as bovine left displaced abomasums (LDA) and human chronic gastritis. However, the effects of ß-HB on stomach injuries have not been explored. To elucidate the possible effects of exogenous ß-HB on the stomach, mice were injected intraperitoneally with ß-HB, and bovine abomasum smooth muscle cells (BSMCs) were treated with different concentrations of ß-HB. We found that ß-HB induced BSMCs endoplasmic reticulum- and mitochondria-mediated apoptotic cell death. ß-HB promoted Bax expression and caspase-12, -9, and -3 activation while blocking Bcl-2 expression. ß-HB also promoted AIF, EndoG release and p53 expression. ß-HB acted on key molecules in the apoptotic cell death pathway and increased p38 and c-June NH2-terminal kinase phosphorylation while inhibiting ERK phosphorylation and PCNA expression. ß-HB upregulated P27 and P21 mRNA levels while downregulating cyclin and CDK mRNA levels, arresting the cell cycle. These results suggest that BSMCs treated with ß-HB can induce oxidative stress, which can be prevented by intracellular calcium chelators BAPTA/AM but not antioxidant NAC. Additionally, these results suggest that ß-HB causes ROS generation through a Ca2+-dependent mechanism and that intracellular Ca2+ levels play a critical role in ß-HB -induced apoptotic cell death. The impact of ß-HB on programmed cell death and oxidative stress in vivo was confirmed in murine experiments. For the first time, we show oxidative stress effects of ß-HB on smooth muscle. We propose that ß-HB is a possible cause of some stomach diseases, including bovine LDA.

Teratogenic effects of diabetic conditions in chick heart in ovo and in micromass culture may be prevented by addition of vitamin C and folic acid.

Maternal diseases like diabetes mellitus may cause developmental defects. Supplementation with folic acid and vitamin C during the periconceptional period has been shown to prevent some neural tube and congenital heart defects. Hearts were dissected from 5 days-old White Leghorn chick embryos, the cells isolated and cultured in micromass under diabetic conditions, with and without folic acid and vitamin C. Contractile activity, cell viability (resazurin reduction) and protein assays were performed. Results indicated diabetic conditions reduced contractile activity and cell viability, whilst vitamin C (100 µM) and folic acid (1 mM) administered concurrently significantly improved them to values comparable with the control. Day 3 chick embryos in ovo were injected with glucose+hydroxybutyrate or a combination of these and vitamins. Diabetic conditions caused gross and histological malformations, but these effects were abrogated by vitamin supplement. Teratogenic effects on heart development could possibly be prevented by vitamin supplementation during pregnancy.

Protein restriction in the pregnant mouse modifies fetal growth and pulmonary development: role of fetal exposure to {beta}-hydroxybutyrate.

Maternal undernutrition during sensitive periods of pregnancy results in offspring predisposed towards the development of a number of diseases of adulthood, including hypertension and diabetes. In order to determine the nature of any gross alterations in fetal growth during early organogenesis, we supplied timed-mated pregnant mice with diets containing 6% protein (6%P), 9% protein (9%P) or 18% protein (18%P; control) from day 0 of pregnancy. At embryonic days 11 (E11), 12 (E12) and 13 (E13), females were killed and fetuses removed. Gross morphological analysis revealed that fetal limb growth was impaired between E11 and E12 in 6%P animals, but this recovered by E13. Likewise, fetal liver growth and lung branching morphogenesis were seen to exhibit an initial growth impairment at E12 followed by a rapid recovery by E13. Coincident with the observed changes in fetal growth, we noted an elevation in maternal hepatic triglyceride content, expression of the ketogenic 3-hydroxy-3-methylglutaryl-CoA synthase 2 (Hmgcs2) and circulating plasma ß-hydroxybutyrate (BOHB). In addition, fetal liver Hmgcs2 expression was switched on by E13 in both 6%P- and 9%P-exposed animals. Exogenous BOHB did not influence branching morphogenesis in fetal lung explant cultures; however, we cannot rule out the possibility that this may occur in vivo. In conclusion, we find that disturbance of fetal growth by maternal dietary protein restriction is associated and therefore potentially indicated by changes in maternal and fetal ketone body metabolism.

In vivo studies of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) based polymers: biodegradation and tissue reactions.

The in vivo tissue reactions and biodegradations of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(lactide) (PLA), poly(3-hydroxybutyrate) (PHB), blends of PHBHHx (X) and poly(ethylene glycol) (PEG) (E) with ratios of 1:1 (E1X1) and 1:5 (E1X5), respectively, were evaluated by subcutaneous implantation in rabbits. Results revealed that the degradation rate increased in the order of PHB < PHBHHx < PLA. During the implantation period, crystallinity of PHBHHx increased from 19% to 22% and then dropped to 14%. Gel permeation chromatography (GPC) displayed increasing polydispersity and typical bimodal distribution from 3 to 6 months. The above results suggested that rapid PHBHHx degradation occurred in amorphous region rather than in crystalline region. While the in vivo hydrolysis of PHB was found to start from a random chain scission both in amorphous and crystalline regions of the polymer matrix, as demonstrated by its hydrolysis process accompanied by a decrease in molecular weight with unimodal distribution and relatively narrow polydispersity. Compared to pure PHBHHx, PHBHHx-PEG blends showed accelerated weight loss of PHBHHx with weak molecular weight reduction. In general, PHBHHx elicited a very mild tissue response during implantation lasting 6 months compared with relative acute immunological reactions observed among PHB and PLA objects, respectively. Pronounced tissue responses were observed in the capsule surrounding E1X1 and E1X5 as characterized by the presence of lymphocytes, eosinophils and vascularization, which might be resulted from the continuous leaching of PEG.

Identification and effects of interaction phytotoxic compounds from exudate of Cistus ladanifer leaves.

Eleven allelochemicals (ferulic acid, cinnamic acid, 4-hydroxybenzoic acid, hydroxycinnamic acid, methyl propionate, oxalic acid, methylmalonic acid, p-anisic acid, butyric acid, 3-hydroxybutyric acid, and azulene) were identified in the exudate of Cistus ladanifer L. We studied the effect of each on germination, cotyledon emergence, root length, and cotyledon length of Rumex crispus. Three groups were distinguished with respect to phytotoxic activity: compounds with low activity (ferulic acid, 4-hydroxybenzoic acid, oxalic acid, methylmalonic acid, p-anisic acid, hydroxybutyric acid, and azulene), with intermediate activity (cinnamic acid and hydroxycinnamic acid), and with high activity (methyl propionate and butyric acid). The effect of the interaction of the compounds was studied. When acting conjointly, all combinations tested produced a more negative effect on both germination and seedling growth than when acting alone. The interaction affected cotyledon emergence and root length more negatively than germination and cotyledon length. When hydroxycinnamic acid and cinnamic acid were added to these mixtures there was an enhancement in the phytotoxic activity, accentuating the effect of the other allelochemicals.